Abstract
Unisite [gamma-32P]ATP hydrolysis was studied in ECF1 from the mutant betaE381C after generating a single disulfide bond between beta and gamma subunits to prevent the rotation of the gamma/epsilon domain. The single beta-gamma cross-link was obtained by removal of the delta subunit from F1 and then treating with CuCl2 as described previously (Aggeler, R., Haughton, M. A., and Capaldi, R. A. (1996) J. Biol. Chem. 270, 9185-9191). The mutant enzyme, betaE381C, had an increased overall rate of unisite hydrolysis of [gamma-32P]ATP compared with the wild type ECF1 due to increases in the rate of ATP binding (k+1), Pi release (k+3), and ADP release (k+4). Release of bound substrate ([gamma-32P]ATP) was also increased in the betaE381C mutant. Cross-linking between Cys-381 and the intrinsic Cys-87 of gamma caused a further increase in the rate of unisite catalysis, mainly by additional effects on nucleotide binding in the high affinity catalytic site (k+1 and k+4). In delta-subunit-free ECF1 from wild type or betaE381C F1, addition of an excess of ATP accelerated unisite catalysis. After cross-linking, unisite catalysis of betaE381C was not enhanced by the cold chase. The covalent linkage of gamma to beta increased the rate of unisite catalysis to that obtained by cold chase of ATP of the noncross-linked enzyme. It is concluded that the conversion of Glu-381 of beta to Cys induces an activated conformation of the high affinity catalytic site with low affinity for substrate and products. This state is stabilized by cross-linking the Cys at beta381 to Cys-87 of gamma. We infer from the data that rotation of the gamma/epsilon rotor in ECF1 is not linked to unisite hydrolysis of ATP at the high affinity catalytic site but to ATP binding to a second or third catalytic site on the enzyme.
Highlights
It had been shown several years ago that the ␥ and ⑀ subunits undergo conformational changes in F1F0 upon membrane energization (4 – 6) and that they shift their positions between the ␣ and  subunits of soluble F1 in response to nucleotide binding [7,8,9]
Unisite catalysis is measured with substoichiometric amounts of [␥-32P]ATP in relation to F1 and is characterized by a highly exergonic ATP binding step (K1), a reversible ATP hydrolysis/synthesis equilibrium that occurs with a negligible change in free energy (K2), and a very slow product release step which is rate-limiting
The kinetics of ATP hydrolysis in a single high affinity catalytic site was measured using [␥-32P]ATP, and the effect of a large excess of ATP as a “cold chase” on this kinetics was studied. This socalled cold chase examines the effect of ATP binding at a second and/or third catalytic site on the kinetics of [␥-32P]ATP hydrolysis in the first, highest affinity site
Summary
It had been shown several years ago that the ␥ and ⑀ subunits undergo conformational changes in F1F0 upon membrane energization (4 – 6) and that they shift their positions between the ␣ and  subunits of soluble F1 in response to nucleotide binding [7,8,9]. To test the accuracy of mixing, gel filtration, and curve fitting methods employed, some rate constants (kϩ1, kϩ3, K2, kϩ2, and k–2) were measured in parallel using the wild type enzyme (Ϯ ␦ subunit) treated in the same conditions used for the E381C ECF1.
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